Because the role of the viral B2
protein in the pathogenesis of nervous
necrosis virus infection remains unknown, the aim of the present study was to determine the effects of B2
protein on
hydrogen peroxide (H2O2)-mediated cell death via mitochondrial targeting. Using a B2 deletion mutant, the B2 mitochondrial targeting
signal sequence ((41)RTFVISAHAA(50)) correlated with mitochondrial
free radical production and cell death in fish cells, embryonic zebrafish, and human
cancer cells.
After treatment of grouper fin cells (GF-1) overexpressing B2
protein with the
anti-oxidant drug,
N-acetylcysteine (NAC), and overexpression of the
antioxidant enzymes, zfCu/Zn
superoxide dismutase (SOD) and zfCatalase, decreased H2O2 production and cell death were observed. To investigate the correlation between B2 cytotoxicity and H2O2 production in vivo, B2 was injected into zebrafish embryos. Cell damage, as assessed by the
acridine orange assay, gradually increased over 24 h post-fertilization, and was accompanied by marked increases in H2O2 production and embryonic death. Increased oxidative stress, as evidenced by the up-regulation of
Mn SOD,
catalase, and Nrf2, was also observed during this period. Finally, B2-induced
dynamin-related
protein 1 (Drp1)-mediated mitochondrial fragmentation and cell death could be reversed by NAC and inhibitors of Drp1 and Mdivi in GF-1 cells. Taken together, betanodavirus B2 induces H2O2 production via targeting the mitochondria, where it inhibits complex II function. H2O2 activates Drp1, resulting in its association with the mitochondria, mitochondrial fission and cell death in vitro and in vivo.